Enzymes in the Environment: Activity, Ecology and Applications - Chapter 16 pptx

16
Hydrolytic Enzyme Activities to Assess
Soil Degradation and Recovery
Tom W. Speir
Institute of Environmental Science and Research, Porirua, New Zealand
Des J. Ross
Landcare Research, Palmerston North, New Zealand
I. INTRODUCTION
A project of the United Nations Environmental Program on Global Assessment of Soil
Degradation concluded, ‘‘Nearly 40% of all agricultural land has been adversely affected
by human-induced soil degradation, and over 6% would require major capital investment
to restore its original productivity’’ (1). It is, therefore, not surprising that, among regula-
tory authorities, there is a strong desire for the development of sensitive indicators to
assess soil degradation. Properties that provide a snapshot assessment of the status of a
soil can determine whether a management practice has had an adverse effect on soil
‘‘health’’ and productivity and, better still, can predict whether a practice will have an
adverse effect if it is continued. This has been one of the major drivers of the worldwide
research effort on soil quality defined as ‘‘the capacity of a soil to function, within ecosys-
tem and land-use boundaries, to sustain biological productivity, maintain environmental
quality, and promote plant and animal health’’ (1). This topic has been the subject of
numerous reviews, such as those found in the Soil Science Society of America Special
Publications 35 (2) and 49 (3). We do not wish to enter the debate concerning a potential
role for enzyme activity measurements in the wide soil-quality context—this topic has
already been reviewed (4–6)—but to focus on the application of soil enzymes to scenarios
in which soil degradation is demonstrable, or at least strongly suspected to be a likely
outcome of a particular land-management practice.
In this review, we do not present the many, sometimes contradictory, reports of effects
of different management practices on soil enzyme activities that have already been reviewed
in detail (6–14) but rather use our knowledge and perceptions of soil enzymes to try to
understand what the enzyme activity measurements are telling us about the soil and how
they can be used to assess soil degradation and recovery. The scenarios we cover are soil

physical degradation as a result of human-induced factors, such as intensive cropping and
soil compaction, and soil loss from mining. In this last example, there is no need to assess
degradation at all; emphasis is on rehabilitation of the land and creation of a productive
Copyright © 2002 Marcel Dekker, Inc.
soil when the mine is closed or moved on across the landscape. We also consider soil
contamination from the dumping or accidental spillage of organic and inorganic materials,
e.g., hydrocarbons and heavy metals, and the application of sewage sludge and pesticides.
II. ENZYMES IN SOIL—OCCURRENCE, LOCATION, AND ASSAY
In order to use soil enzyme activity measurements to provide information that will enable
us to assess the extent of soil degradation or recovery, we need to recognize the limitations
of our methodology and our knowledge of the role and function of soil enzymes.
Because of the diversity of life in the soil, it is probable that most known enzymes
could be found in a soil sample. However, the activities that have been measured are
limited to a few oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), and lyases
(EC 4) (11). It is impossible to extract a significant proportion of any enzyme activity
from soil, unlike other living systems, and activities are therefore invariably assayed in
situ. It is, consequently, not possible to assign activity to individual organisms or even
to particular groups of organisms. The enzyme activity measured represents the sum of
contributions from a broad spectrum of soil organisms (including plants) and also extracel-
lular or abiontic enzymes (15,16) that retain their activities away from the living cell. For
enzymes that do not require cofactors and that are not components of catabolic or anabolic
sequences, a significant proportion of the total activity may be extracellular and any cata-
lytic function performed by these particular enzymes is purely opportunistic. This does
not mean that soil organisms are unable to take advantage of this catalysis, and it may
be that such enzymes play an important role in the initial degradation of macromolecular
substrates in soil (17,18). The most studied group of soil enzymes that are likely to have
a significant active extracellular component are the hydrolases; it is generally accepted that
these enzymes comprise a metabolically vital intracellular fraction and an opportunistically
active extracellular fraction divided among several locations in the soil (19). The propor-
tional size of this extracellular component is generally unknown and probably varies from

enzyme to enzyme.
Most hydrolases are investigated by using artificial substrates and assay conditions
that are quite foreign to those prevailing in soil. Substrates are usually small molecules,
often simple esters combining the functional group of the substrate, e.g., phosphate (for
phosphatase) or glucose (for β-glucosidase) with a chromophore, such as p-nitrophenol,
for ease of extraction from soil and ease of assay. Activity normally is measured under
buffered conditions at the optimal pH for the enzyme, at enzyme-saturating substrate con-
centrations, and usually at a temperature substantially greater than would generally prevail
in soil (20). The composition and molarity of the buffer are especially important, because
a buffer found suitable for some soils is not necessarily suitable for others (21). For exam-
ple, a commonly used buffer (acetate-phosphate) for assays of invertase activity inhibited
activity in acid grassland soils and could thereby have obscured relationships of invertase
with other soil and environmental factors (22).
Under suitable assay conditions, the measured activity of an enzyme such as phos-
phatase, for example, represents only the potential p-nitrophenyl phosphate-hydrolyzing
capacity of the soil. It is probable that not all of the numerous phosphatases present are
assayed (all may not be active against this substrate), and it is certain that the reaction
rate would be much greater than the rate of phosphate production from organic phosphorus
compounds in the unamended soil. It is, therefore, difficult to see how a direct causal role
in the phosphorus fertility of a soil can be ascribed to the conglomerate of phosphatase
Copyright © 2002 Marcel Dekker, Inc.
enzymes assayed in this way. Of the hydrolases, only urease and invertase are measured by
using their natural substrates, viz., urea and sucrose, respectively. However, the artificial
conditions used in the assay of these enzymes again preclude any direct connection be-
tween measured activity and substrate hydrolysis that occurs naturally in soil. Although
starch and cellulose are used as substrates for amylase and cellulase, the chemical forms
and purity of these substrates would be very different from those found in soil.
One enzyme that has been studied extensively because of its perceived close relation-
ship with microbial activity is the oxidoreductase dehydrogenase. This enzyme, or group
of enzymes, is a component of the electron transport system of oxygen metabolism and

requires the organization of the living intracellular environment to express its activity.
Consequently, dehydrogenase activity is not likely to be present in any of the extracellular
compartments occupied by the hydrolases. The absence of an extracellular component
means that dehydrogenase activity may not be well suited to assess soil degradation be-
cause it is likely to fluctuate, as does microbial activity, in response to recent management
and/or seasonal (climatic) effects (5). Although the presence of dehydrogenase activity
in soil should reflect the activity of physiologically active microorganisms, including bac-
teria and fungi (23), measured dehydrogenase activity does not correlate consistently with
microbial activity (6). There are several possible reasons for this, including unsuitable
assay conditions, the presence of extracellular phenol oxidases, and the presence of alterna-
tives to the added electron acceptor (substrate) (6). These electron acceptors may be com-
mon soil constituents, such as nitrate (24) or humic acids (23). It also has been found that
Cu reduces apparent dehydrogenase activity, not by inhibiting the enzyme, but by interfer-
ing with the assay procedure (25). These procedural artifacts raise questions about the
accuracy of dehydrogenase activity results, especially in situations in which a management
practice may be changing the amount of a soil component or adding a xenobiotic chemical
that may interfere with the enzyme assay. In view of these concerns, and the likely suscep-
tibility of dehydrogenase activity to transitory fluctuations, we focus only on the hydrolase
enzymes in this review.
Obviously, at least a component of every soil enzyme has a vital metabolic role in
situ, but it is most unlikely that any indication of the role(s) or even the real activity of
the enzyme(s) under field conditions can be gained from the assay methods used. The
assertion of Skujins that ‘‘obtaining a fertility index by the use of abiontic soil enzyme
activity values seems unlikely’’ (19) applies as much today as it did over 20 years ago.
It is important that these considerations be acknowledged when investigating how enzyme
activities can be used to assess soil degradation and recovery.
III. SOIL HYDROLASE ENZYMES TO ASSESS PHYSICAL
DEGRADATION OF SOILS
Soil degradation through loss of organic matter and structural integrity is a well known
outcome of an intensive cropping regime. There have been many studies comparing the

chemical, physical, and biological properties of soils subjected to conventional cultivation
practices with those subjected to minimal or no tillage. When comparing conventionally
ploughed and no tillage plots, Klein and Koths (26) found that urease, protease, and phos-
phatase activities were higher under no tillage than under ploughed treatments. Dick (27)
observed the same results with acid phosphatase, arylsulfatase, invertase, amidase, and
urease in the top 7.5 cm of soil and concluded that changes in activity were not attributable
to long-term pesticide application. Gupta et al. (28,29) compared soils that had been under
Copyright © 2002 Marcel Dekker, Inc.
cultivationforupto80yearsandfoundthattheirarylsulfataseandphosphataseactivities
wereconsiderablyreducedwhencomparedwiththoseinnative,uncultivatedsoils.Culti-
vationdecreasedtheenzymeactivitiesinallaggregatesizefractionsofa69-yearcultivated
soilanddecreasedtheMichaelisconstant(K
m
)andmaximumreactionrate(V
m
)forarylsul-
fataseinallcultivatedsoils.Theauthorsconcludedthatdecreasedarylsulfataseactivity
inthecultivatedsoilsreflected‘‘thereductionsinorganicmattercontentandmicrobial
biomassandactivityofthesoilassociatedwithlandmanagement’’(29).Theyalsopro-
posedthatclearingandcultivationofnativesoilsresultinnativesoilorganicmatterbeing
transformedintomoreinertformsthatarelesslikelytoformcomplexeswitheitherthe
enzymeoritssubstrate;thiswouldaccountforincreaseinsubstrateaffinity(i.e.,lower
K
m
)inthecultivatedsoils.Changesinenzymeactivitiesindifferentaggregatesizefrac-
tionsundercultivationregimesalsohavebeenobservedbyKandelerandassociates(30).
Theeffectsofthreedifferenttillagesystemsonthetotalxylanase,invertase,andalkaline
phosphataseactivitiesofthe0-to10-cmlayerofsoilandalsoontheproportionsfound
indifferentparticlesizefractionsareillustratedinFig.1.Theauthorsalsofoundthatthe
reduced tillage and, especially, the conventional tillage treatments had decreased soil or-

ganic C content in the coarsest (Ͼ200-µm) fraction. This would have been the principal
reason for the greatly reduced soil xylanase activity in the conventional tillage treatment,
because a large proportion of this enzyme activity was located in this coarsely textured
organic fraction (Fig. 1). The other two enzymes, invertase and alkaline phosphatase, were
more closely aligned with the finer soil fractions and were less affected by tillage, although
the proportions in the coarsest soil fraction also were diminished (30).
Dick et al. (31) examined skid-trail soils, i.e., soils compacted by dragging logs from
forestry operations, and found that compacted soils had considerably lower phosphatase,
arylsulfatase, and dehydrogenase activities than the control soil, especially in the subsoil.
They also showed there was a very strong correlation between the enzyme activities and
soil organic C and microbial C. They concluded that a combination of physical factors
and impaired root growth was the probable reason for these compaction effects.
Sulfatase activity in arctic tundra soils also was lowered significantly after vehicle
disturbance (32). The wetter, depressed portions of the vehicle tracks supported more
vigorous plant growth as a result of nutrient influx caused by the channeled water flow.
Sulfatase activity levels in these wet areas were considered to have become depressed
because of end product inhibition or inhibition by other ions, e.g., phosphate.
Apart from the previous example, usually the main result of these, and the many
other studies (33–35), is that soil enzyme activities decline in proportion to the loss of
soil organic matter. This tendency does not provide any more information about potential
soil degradation under a cropping regime than does the measurement of organic C alone
or any information about the short-term productivity of the soil. An intensively cropped
soil with lower enzyme activity and organic matter content than those of a neighboring
native soil may, in fact, be far more productive because it has greater nutrient status. Many
studies over the years have shown that, under intensive agriculture, in which nutrients can
be added from a bag, soil enzyme activities are not good predictors of soil fertility and
productivity. However, it is also generally recognized that such intensive cropping prac-
tices are not sustainable in the long term and that the soils become much more prone
to erosion, waterlogging, and compaction. Residue-management trials have shown that
conservation tillage and organic-residue-amendment strategies maintain soil organic mat-

ter and retain soil physical characteristics (26,33,36–38). Therefore, if a soil enzyme can
tell us something about the location and perhaps the quality of soil organic matter in
Copyright © 2002 Marcel Dekker, Inc.
Figure 1 Xylanase (a), invertase (b), and alkaline phosphatase (c) activities in particle-size fractions
of the 0- to 10-cm layer of a Haplic Chernozem soil. (Adapted, with permission, from Ref. 30.)
cropped soils, e.g., by measurement of xylanase activity in soil particle-size fractions (30),
then it may be possible to use its activity as an early warning of potential structural degra-
dation. Changes in enzyme kinetic properties, if they reflect changes in organic matter
quality (29), also may provide more information about the status of a soil than can be
gained from its organic matter content.
IV. SOIL HYDROLASE ENZYMES TO ASSESS SOIL RECOVERY AND
DEVELOPMENT AFTER MINING
Many studies have demonstrated the decline of organic C, microbial biomass, and enzyme
activities with increasing soil depth. Ross et al. (39) showed the removal of 10 cm, and
especially 20 cm, of topsoil from temperate pasture plots markedly lowered activities of
a number of enzymes. This finding is not at all surprising, since the top centimetres of a soil
are the major loci of biological activity and organic matter. What is especially interesting,
however, is that removal of 10 cm of topsoil from this pasture resulted in a new topsoil
with approximately 40% less organic C, but more than 60% lower urease and phosphatase
Copyright © 2002 Marcel Dekker, Inc.
activities, 75% lower invertase and amylase activities, and more than 80% lower cellulase
and xylanase activities; only sulfatase matched organic C with a 40% decline in activity
(39). Speir et al. (40) showed that organic C declined relatively linearly with depth in a
pasture soil, whereas most enzyme activities and soil respiratory activity and microbial
biomass fell much more rapidly in the top 15 cm than in the remainder of the soil profile
(Fig 2). Here again, sulfatase activity most closely matched the decline of organic C. It
Figure 2 Influence of depth on soil chemical properties and enzyme activities. (Adapted, with
permission, Ref. 40.)
Copyright © 2002 Marcel Dekker, Inc.
wasconcludedthatthecarbohydraseenzymes(amylase,cellulase,invertase,xylanase,

and,toalesserextent,xylopyranosidase)maybecloselyrelatedtocurrentsoilbiological
activityandbedisproportionatelyhigherthanpredictedfromorganicCcontent,inthe
topmostsoillayer,becauseofimprovedaerationandsubstrateavailability(40).Onthe
otherhand,urease,phosphatase,andespeciallysulfatasemaybemorerelatedtototal
organicCbecauseoftheirstabilized,extracellular,organomineral-boundcomponent.
Technologiestorecoversuchsoilsafterminingandmethodstoassesstheirrecovery
areequallyapplicabletothedevelopmentoflandscapesreconstructedafterunderground,
strip,oropencastminingforcoalandothermineralresources.Itisestimatedthatabout
1600ϫ10
9
m
3
ofminespoilshadaccumulatedontheEarth’ssurfaceupto1980and
hadincreasedbyabout40ϫ10
9
m
3
peryearby1998(14).Rehabilitationofthesespoils
anddegradedlandscapesisnowanintegralpartofminingoperationsinmanypartsof
theworld.Theenzymologicalcharacteristicsoftheseconstructedor‘‘technogenic’’soils
havebeenextensivelyreviewed(7,11,14,41,42).
Technogenicsoilsmayhavea‘‘topsoil’’composedofentirelysubsurfacematerials
orthestockpiledoriginaltopsoilorsomeintermediatecombination.Stockpilingoftopsoil
leadstoadeclineofsoilbiologicalactivity(14),presumablyduetothelackofreplen-
ishmentofreadilydegradableplantresiduesandtofactorssuchascompactionandreduced
aeration.Speiretal.(43)foundthattheprotease,sulfatase,andureaseactivitiesof12
soilsleftfallowinapottrialdeclinedmarkedlyover5months,whereasactivitiesgenerally
remainedunchangedorincreasedifthesoilswereplantedwithperennialryegrass.The
declineinthefallowtreatmentswasprobablyattributabletodecliningmicrobialactivityas
plantresiduesweredegraded,leavingonlymoreintransigentorganicmatter.Itisprobable,

therefore,thattheinitialbiologicalactivityofthetopsoilofatechnogenicsoil,nomatter
howitisconstructed,isconsiderablylowerthanthatoftheoriginalsoilonthesite.It
certainlydoesnothavethehighbiologicalandenzymaticactivitiesfoundinthevery
surfacelayerofanundisturbedsoil(40)(Fig.2).
DickandTabatabai(9)concludedthat‘‘inenvironmentsinitiallydevoidofplant
ormicrobiallife,asisoftenfoundfordrasticallydisturbedlands,aclosecorrelationexists
betweenplantandmicrobialcommunitiesandtheexpressionofenzymeactivities.’’
Therefore,intheearlystagesofrecoveryoflandthathashadthesurfacesoilremoved
(e.g.,aftererosionortopsoilmining),orintheearlystagesofdevelopmentoftechnogenic
soilsfromstockpiledsoilandoverburdenmaterials(e.g.,landreclamationaftermining),
acloserelationshipbetweenplantproductivityandsoilenzymeactivitymightbe
expected.
Rossetal.(39,44)investigatedtherelationshipbetweenrecoveryofsoilbiochemical
propertiesandplantproductivityinatemperatepasturesoilthathadhad10cmor20cm
oftopsoilremovedinatrialtosimulatetheeffectsoftopsoilmining.Theratesofrecovery
ofinvertase,amylase,cellulase,andxylanase,butnotphosphatase,sulfatase,orurease,
were,after3years,muchgreaterthantherateofrecoveryoforganicC(Table1).However,
after 5 years, the recovery of all properties had slowed. During the early stages of restora-
tion, the enzyme activities generally correlated very closely with pasture production, but
in the longer term (5 years) the activities were more closely related to the recovery of
organic C (Table 1). The comparatively rapid recovery of invertase activity also occurred
in a temperate hill pasture (45) where the original soil had eroded in slips of up to 60-
cm depth. Restoration of invertase activity in regenerating pasture was complete within
11 years, whereas phosphatase activity was then only about 36% of that of uneroded
topsoil (DJ Ross, TW Speir, AW West, personal communication, 1984).
Copyright © 2002 Marcel Dekker, Inc.
Table 1 Recovery of Organic C and Enzyme Activities, and Their Correlation with Herbage
(Pasture Grasses and Clover) Production, in Soil Stripped of 20 cm of Topsoil
Percentage of control (unstripped) Correlation with herbage
soil value after production, all data up to

Property 0.5 year 3 years 5 years 3 years 5 years
Organic C 41 59 66 0.68* 0.41
Urease 16 46 61 0.79** 0.73**
Invertase 29 88 88 0.92*** 0.51*
Amylase 34 84 101 0.70* 0.40
Cellulase 32 84 82 0.90*** 0.63**
Xylanase 12 70 80 0.91*** 0.71***
Phosphatase 40 61 76 0.90*** 0.46
Sulfatase 19 48 62 0.91*** 0.47*
*, **, *** ϭ P Ͻ 0.05, 0.01, 0.001, respectively.
Source: Adapted from Refs. 39 and 44.
In an investigation of different replacement strategies in the construction of techno-
genic soils after simulated lignite mining, herbage yields in all replacement treatments
reached the level of the temperate pasture control plots within 3 years, as long as the soil
was ripped to alleviate compaction (46). Biochemical activities, including those of in-
vertase and sulfatase, increased rapidly in all treatments in the early stages of the trial.
Invertase activity reached the level of the control soil after 3 years, and sulfatase attained
that level in two of the three replacement treatments after 5 years. In contrast, organic C
content had increased linearly from 47% to 76% of that of the control at the start of the
trial to 68%–92% after 5 years. The correlations of organic C and invertase and sulfatase
activities with herbage yields, using all data over the 5 years of the trial, are shown in
Table 2. The levels of soil invertase activity and, to a lesser extent, sulfatase activity
provided a good indication of herbage production as restoration progressed. It was con-
cluded that plant materials would have contributed appreciably to the rapid increase of
Table 2 Correlations of Soil Organic C and
Invertase and Sulfatase Activities with Pasture
Herbage Yields from Technogenic Soils
Constructed Using Three Soil Replacement
Strategies, Including All Data over the 5 Years
of the Trial

Soil replacement treatment
Property 1
a
23
Organic C 0.20 0.31 0.39
Invertase 0.59** 0.75*** 0.55**
Sulfatase 0.37 0.59** 0.77***
a
Treatments were 1, horizon A/B/C; 2, (A ϩ B)/C; 3,
(A ϩ lignite overburden (O))/B ϩ O)/C ϩ O).
**, *** ϭ P Ͻ 0.01, 0.001, respectively.
Source: Adapted from Ref. 46.
Copyright © 2002 Marcel Dekker, Inc.
soil invertase activity. Such a rapid buildup of soil biological activity and of plant pro-
ductivity is the exception rather than the rule. Most investigations have shown that the
enzyme activities of technogenic soils generally were considerably lower than those of
control or native soils, even after 20 or more years (11,14). It is likely that optimization
of factors, such as fertilizer inputs, soil aeration, drainage, and bulk density, as well as
climate, resulted in extremely favorable conditions for soil recovery in the New Zealand
study (46).
It is interesting to speculate why there is a strong relationship between plant produc-
tivity and soil enzyme activity in at least the early stages of development of a fertile soil.
Plants and nutrients in the soil are the drivers of the recovery, as plants provide C to
enable the initially sparse microbial populations to proliferate. The microorganisms and,
to a lesser extent, the plant fragments are the principal source of the enzymes. Both intra-
cellular and extracellular enzyme concentrations increase in proportion to microbial num-
bers, and the extracellular enzymes are able to become bound and stabilized at the many
unoccupied binding sites in the soil. As already mentioned, it is possible that an initial
buildup of an extracellular enzyme component is vital during the early stages of microbial
proliferation, because such enzymes may catalyze the commencement of degradation of

the macromolecular plant substrates (17,18). Once these mechanisms are under way, it
might be expected that the rate of recovery of soil enzyme activity would match that of
plant productivity and be proportional to the input of plant residues. If nutrients and physi-
cal conditions are not limiting, plant productivity drives the process toward the levels of
biological activity found in nearby undisturbed soils with the same parent materials and
chemical properties. The rate of recovery of biological and enzyme activities exceeds the
rate of recovery of soil organic matter content. As time passes and the sites for stabilization
of extracellular enzymes become saturated, their concentrations may level off, and in-
creases in enzyme activity with increasing microbial numbers and organic matter content
may then be a function of intracellular enzymes only (microbial and plant). If the soil
nutrient status and physical status are not limiting, plant productivity may still drive in-
creased microbial numbers and organic matter content but may no longer be related di-
rectly to total soil enzyme activity.
In soil-recovery situations, such as those described, the enzyme activities do not
necessarily need to be assigned a role in the recovery process. They are merely indicators
that can be used to give progress reports on the rate of recovery of plant productivity and
perhaps predict how long full recovery will take. Some are better indicators than others;
this may be a function of the enzymes themselves or it may be specific to a site, or soil,
or particular vegetation. The carbohydrase enzymes, especially those involved in the
breakdown of macromolecular plant residues (e.g., xylanase), or invertase because of its
relationship with plant materials (47), may be better predictors than the more often assayed
phosphatase, sulfatase, and urease enzymes. As time progresses, the activities of this latter
group are probably more closely related to the soil organomineral components, and their
(presumably) large, stabilized, extracellular component mask more subtle changes re-
sulting from increasing microbial and plant production. Overall, however, we do not fully
understand these relationships. Therefore, predictions of productivity or recovery rates in
degraded or technogenic soils from the assay of a single soil enzyme, or even of several
enzymes in isolation from other soil properties, would be unwise; at this stage, a predictive
role for enzymes in soil recovery is still an experimental tool.
Another approach to predicting the effects of disturbance and the success of soil

rehabilitation procedures has been to use a multivariate analysis technique (48). This
Copyright © 2002 Marcel Dekker, Inc.
method uses biological properties, including the enzymes alkaline phosphatase, sulfatase,
arginine deaminase, protease, invertase, and dehydrogenase, in combination with other
soil properties and is able to discriminate between soils affected by oil well drilling, surface
mining, hydrocarbon spills, and pipeline construction, and undisturbed soils from similar
areas. Although the reason for the choice of these particular enzymes is not clear, a dis-
criminant function combining seven properties, including alkaline phosphatase and argi-
nine deaminase activities, correctly classified 86% of the undisturbed soils and 70% of
the disturbed soils. This investigation, which comprised 68 soils covering five Canadian
soil groups, appears to provide a basis for reclassifying a once-disturbed soil as having
been remediated sufficiently to be equivalent to an undisturbed soil.
V. SOIL HYDROLASE ENZYMES TO ASSESS
SOIL CONTAMINATION
A. Contamination by Crude Oil and Oil By-Products
Because of the huge volumes of oil and its by-products that are produced, transported,
and stored, there is a very serious threat of soil contamination in the vicinity of oil fields,
refineries, and storage and distribution facilities. The effects of oil pollution on the activi-
ties of soil enzymes have been extensively reviewed by Kiss et al. (14). We therefore
give only a synopsis of the data presented in that review and limit our discussion to the
interaction of oil products with enzymes and the capacity of enzyme activity measurements
to ascertain the extent of soil degradation that has occurred.
Polar organic solvents such as ethanol and acetone destroy enzyme activity by pro-
tein denaturation. However, nonpolar organic compounds, such as hydrocarbons, are hy-
drophobic and do not interact significantly with proteins in solution. In soil, crude oil and
some of the heavier oil fractions, if present in very high concentrations, may block the
expression of enzyme activity by coating organomineral and cell surfaces and thereby
prevent soluble substrates reaching the enzyme molecules. It may be concluded that the
lighter petroleum products are not particularly inhibitory toward soil enzymes because of
the extensive use of toluene, at concentrations up to 25% of the assay volume (19), as a

microbial inhibitor in soil enzyme assays.
In the research reviewed by Kiss et al. (14), large amounts of crude oil were required
to cause a significant reduction of soil enzyme activities, with concentrations as high as
100 kg m
Ϫ2
reducing invertase, protease, and phosphatase activities by 54%, 62%, and
50%, respectively (49). Although the activity of most soil enzymes is adversely affected
by crude oil, urease activity often increases (14). Different responses to crude oil were
also observed in another study (50); cellulase activity declined whereas aryl-hydrocarbon
hydroxylase activity increased; a shift in catabolic activity of the soil microbiota in re-
sponse to the new carbon source is indicated. Important findings of Samsova et al. (51)
were reduction of protease activity, increase in urease activity, and death of all plants on
contamination with 8% crude oil. Other studies have shown that at moderate levels of
oil contamination, some enzyme activities declined and some increased, most microbial
populations increased, but plant growth was usually impaired (14). It would seem, there-
fore, that soil enzyme activities are less sensitive than plants to soil degradation by crude
oil. In some instances, however, they may provide information about the potential for the
soil microorganisms to metabolize the oil and for the contaminated soil to recover from
the pollution.
Copyright © 2002 Marcel Dekker, Inc.
Although toluene is not particularly inhibitory to soil hydrolase activities, refined
oils can inhibit urease activity. In three soils, inhibition increased in the order kerosene Ͻ
diesel Ͻ motor oil Ͻ leaded gasoline, at amendment concentrations of 5%, 10%, and 25%
(w/w), but only leaded gasoline at 25% resulted in more than 50% loss of urease activity
(52). Amendment of soil with jet fuel at rates of 5% and 13.5% reduced the rate of fluores-
cein diacetate (FDA) hydrolysis (esterase activity) (53). However, if the soil was subjected
to a bioremediation treatment (lime, fertilizers, and simulated tillage), FDA hydrolysis
increased rapidly and markedly after a 1-week lag period. The reduced activity in the
nonremediated soil was attributed to inhibition by jet-fuel degradation products. Inhibition
by these fuel products may be caused by the aromatic, and not the aliphatic, components

of the hydrocarbon mixtures, and possibly only by benzene (54–56).
B. Contamination by Heavy Metals and Metalloids
1. Inhibitory Effects of Heavy Metals in Soil
Heavy metals are toxic to living organisms primarily because of their protein-binding
capacity and hence their ability to inhibit enzymes. The cationic metals are noncompetitive
inhibitors, which bind irreversibly with sulfydryl and carboxylate groups and with histi-
dine, altering protein structure and the conformation and accessibility of the enzymes’
active sites. The anions of metals and metalloids, e.g., As[V], W[VI], and Mo[VI], may
have analogous structures to products and/or inhibitors of certain enzymes and are, there-
fore, likely to be competitive inhibitors. For example, the inhibition of soil phosphatase
by HAsO
4
2Ϫ
,WO
4
2Ϫ
, and MoO
4
2Ϫ
has been attributed to the structural similarity of these
anions to HPO
4
2Ϫ
(or H
2
PO
4
Ϫ
), the product and also an inhibitor of this enzyme’s activity
(57). Similarly, these anions inhibit sulfatase because HPO

4
2Ϫ
/H
2
PO
4
Ϫ
also inhibit this
enzyme (58).
In solution, cationic metal salts are effective enzyme inhibitors at very low concen-
trations. However, the many metal-amendment studies (e.g., 59–65) and field studies at
contaminated sites (e.g., 66–70) have shown that inhibition of soil enzymes usually re-
quires much higher heavy-metal concentrations. There are two possible explanations for
this behavior:
1. The physical surroundings of the soil enzymes protect them from exposure to
the metals.
2. The metals are rendered less available to the enzymes by interaction with soil
constituents.
The first mechanism is possible for intracellular enzymes, via mechanisms that pre-
vent metals from passing through cell membranes. Protective mechanisms for extracellular
enzymes appear less likely since metal ions are smaller than most enzyme substrates.
However, extracellular enzymes can be protected if the site of inhibition is remote from
the enzyme’s active site and is inaccessible to the metal ion. The second mechanism is a
certainty. Heavy metals interact very strongly with soil inorganic and organic constituents
through adsorption, chelation, and precipitation reactions that render them much less avail-
able. Effectively, most of the metal is ‘‘locked-up,’’ and only the small amount in soluble
form at the site of enzyme activity (intracellular or extracellular) is able to interact with
the enzymes. La
¨
hdesma

¨
ki and Piispanen (71), using fractionation techniques, found a very
much greater inhibitory effect of Zn and Cu salts on protease, cellulase, and amylase
Copyright © 2002 Marcel Dekker, Inc.
activities in fractions from which the clay and humus colloids had been separated out
than in the original soil. One or both of the mechanisms could account for this increased
inhibition.
The capacity of a soil to protect its enzymes from inhibition by heavy metals is,
therefore, a function of its ability to lock up the metals; therefore, there should be a rela-
tionship of soil texture and organic matter content with enzyme inhibition. In support of
this premise, it has been shown that heavy metals caused greater inhibition of enzyme
activities and other biochemical properties in coarse-textured soils than in fine-textured
soils (72–78). This also can be seen in the data of Tabatabai et al. (57–60) and has been
attributed to the lower surface area, lower cation exchange capacity (CEC), and generally
lower organic matter content of these coarse-textured soils, all of which diminish their
capacity to reduce the solubility of metal ions (72). Inhibition of enzyme activity in heavy-
metal-contaminated soil should, then, reflect the ‘‘bioavailability’’ of the metals, since
the mechanisms that are protecting soil enzymes are likely to be the same mechanisms
limiting metal uptake by plants and soil organisms. Therefore, soil enzyme activity may
be considered a surrogate measurement of the impact of metals on soil biota as a whole
or of their uptake by, and their toxicity to, plants. Use of an enzyme activity to assess
soil degradation by heavy metals requires no knowledge of what the enzyme is doing in
the soil; it is merely an indicator or biosensor of a more general effect.
2. Dose–Response Models
This potential ecotoxicological role for soil enzymes has been investigated in several stud-
ies to determine an ecological equivalent of LD
50
, viz, the ecological dose 50%, ED
50
,of

heavy metal in soil. ED
50
is defined as the concentration of a toxicant that inhibits a
microbially mediated ecological process by 50% (79).
Haanstra and associates (80) developed a ‘‘logistic response model’’ to describe the
observed sigmoidal relationship between biological activity (in this instance, respiration)
and the natural logarithm of the toxicant (Ni) concentration (Fig 3). Although ED
50
de-
termined from this model was found to be a useful measure of toxicity, it provided no
Figure 3 The logistic response curve and the relationship describing it. Parameter Y, enzyme
activity; X, natural logarithm of the heavy metal concentration; c, uninhibited enzyme activity;
b, slope parameter indicating the inhibition rate and equal to 4.39/(0.1c Ϫ 0.9c); a, logarithm of
concentration at which enzyme activity is half the uninhibited level (a ϭ 0.5c); E, stochastic error
term. (With permission from Ref. 74.)
Copyright © 2002 Marcel Dekker, Inc.
information about the ‘‘suddenness’’ of the decrease in activity (80). For this reason, a
further measure, the ecological dose range (EDR), defined as the dose range over which
activity decreases from 90% to 10% of the undisturbed activity, was proposed. Haans-
tra and coworkers used this approach to determine ED
50
values and the EDR ranges for
urease, phosphatase, and arylsulfatase activities in five soils 6 weeks and 18 months
after amendment with six heavy metals (73–75). Generally, ED
50
values were predictably
lower in soils with low CEC and organic C content, e.g., sandy soils. ED
50
values usually
were lower after 18 months than after 6 weeks, although few of the differences were

significant. These studies indicated that considerable enzyme inhibition could be expected
at soil metal concentrations that were then considered acceptable under existing legislation
(74,75).
More recently Speir et al. (76,77) used two Michaelis–Menten enzyme-inhibition
kinetic models, in place of this sigmoidal dose–response model, to determine ED
50
values
for the inhibition of soil enzyme activities and other biological properties by heavy metals.
There were two principal reasons for this different approach.
1. These Michaelis–Menten models have a physical interpretation: i.e., they can
explain the behavior of an enzyme exposed to an inhibitor.
2. The logarithmic relationship represented by the sigmoidal dose–response curve
is elongated to an exaggerated extent at low inhibitor concentration. Because
it is not possible to fit a zero concentration to a logarithmic curve, an arbitrary
value of 10
Ϫ3
mg kg
Ϫ1
metal (73,74) or 10
Ϫ3
mmol kg
Ϫ1
metal (75) was assigned
to the unamended soil. In many instances, the lowest amendment concentration
lay well beyond this initial part of the curve.
The first Michaelis–Menten model describes full enzyme inhibition, i.e., fully com-
petitive, fully noncompetitive, and prescribes a linear relationship between the reciprocal
of reaction rate (v) and inhibitor concentration (i) (76,77,81). This model gives a hyper-
bolic relationship between reaction rate and inhibitor concentration, and ED
50

is the con-
centration resulting in a 50% loss of activity (Fig. 4). The second model describes partial
inhibition, i.e., partially competitive, partially noncompetitive (Fig. 4). In both instances
Figure 4 Relationship between reaction rate (v) and heavy metal concentration (i) as described by
the full-(—) and partial-(––) inhibition models. Parameters: c
1
and c
2
, ED
50(1)
, and ED
50(2)
represent
uninhibited rates and ED
50
values for the full- and partial-inhibition models, respectively; c
2
a/b,
minimum (asymptote) for the partial-inhibition model. (With permission from Ref. 76.)
Copyright © 2002 Marcel Dekker, Inc.
allconstants,a,b,andc,arealwayspositiveandbϾc.Inthesecond,partial-inhibi-
tion,model,theinhibitorreducestheaffinityoftheenzymeforitssubstratebutdoes
notpreventtheenzyme-catalyzedreaction.Astheinhibitorcombineswiththeenzyme,
inhibitionincreasestoadefinitelimitbeyondwhichincreasinginhibitorconcentration
hasnofurthereffect.Therefore,themodeldescribesahyperbolicrelationshipinwhich
activityfallstoanasymptoticvalueasinhibitorconcentrationincreases(Fig.4).Ifthis
asymptote occurs at above 50% inhibition, a true ED
50
cannot be estimated. We can,
however, redefine ED

50
in this situation, as the inhibitor concentration that results in the
loss of 50% of all of the activity that can be lost, i.e., a fall to 50% of the asymptote
activity value (Fig. 4).
To date, this Michaelis–Menten technique has been used for only three contrasting
soils and two ‘‘heavy metal’’ species—hexavalent Cr and the metalloid As in its pentava-
lent oxidation state. Cr[VI] was shown to be a potent inhibitor of soil phosphatase and
sulfatase activities, especially in a coarse-textured sandy soil (phosphatase ED
50
of 0.078
mmol kg
Ϫ1
(4 mg kg
Ϫ1
), sulfatase ED
50
of 0.2 mmol kg
Ϫ1
(10 mg kg
Ϫ1
), but a less potent
inhibitor of urease activity (76). As[V], in contrast, was only a moderate inhibitor of
phosphatase and sulfatase activities and was ineffective against urease (77). In almost
every instance in which the inhibition data fitted both models, the second model provided
the better fit, implying that the inhibition was partial.
3. Interpretation of Dose–Response Data
There are at least five points that need to be considered when interpreting data derived
from such dose–response curves. (1) The first concerns the actual significance of ED
50
values. If the enzyme responses are truly indicative of effects on soil organisms, then 50%

loss of activity may well be unacceptable. These models do allow determination of the
heavy metal concentrations causing significantly less than 50% inhibition, e.g., ED
10
. The
current trend in assessment of environmental effects of contaminants is to find the lowest
observed adverse effect concentration (LOAEC) (82,83), which would obviously be much
less than ED
50
.
(2) How should ED
50
values be interpreted when the inhibitor causes activity to fall
to an asymptotic value, as predicted by the partial inhibition model (Fig. 4)? Speir and
colleagues (76,77) showed that in a coarse-textured sandy soil phosphatase and sulfatase
activities were reduced by only about 40% by Cr[VI] and As[V], respectively. However,
in both instances, the excellent fit to model 2 indicated that most of this inhibition occurred
at relatively low inhibitor concentrations. If this is partial inhibition, then all the inhibitor
does is rapidly reduce the affinity of enzyme for its substrate. The reaction continues, but
at a lower rate, which may not be particularly detrimental. However, in the complex soil
medium, this situation also could be explained by complete inhibition of a sensitive com-
ponent of the enzyme. If this happened to be the intracellular component, with the extracel-
lular part possibly being protected from inhibition by its physical location, the conse-
quences could be much more serious and indicate a potentially severe impact on the soil
microbial population.
(3) Results to date (73–77) have revealed that soil enzymes are affected differently
by different metals and respond differently in different soils. This makes it difficult to
decide what enzyme(s) should be used as indicator(s). Since a component of every soil
enzyme has a metabolic role in soil organisms, we probably should choose enzymes that
are particularly sensitive to the metal in question. For example, phosphatase was moder-
ately inhibited by As[V], but urease was unaffected (58,77). This is because arsenate is

Copyright © 2002 Marcel Dekker, Inc.
a structural analog of phosphate, a known feedback inhibitor of phosphatase. On the other
hand, urease was particularly sensitive to cationic forms of heavy metals (58), presumably
because of the presence of sulfydryl groups in the vicinity of its active site.
(4) The experiments used to derive these dose–response relationships are very arti-
ficial and do not reflect how metals enter soil, except in a rare chemical-spill situation.
Giller and associates (84) stated that this experimental approach is simplistic in that it
bears little relationship to most ‘‘real-life’’ contamination of soils, in which metal concen-
trations are built up over many years and the metals are well equilibrated with the sur-
rounding soil. In reality, metals generally are applied to soils in relatively small doses
and often are bound strongly in organomineral complexes, e.g., in sewage sludge. In spite
of this artificiality, however, the results from early amendment studies with metal salts
(e.g., 57–64,73–75) have been used as supporting information to derive limits, based on
LOAEC principles for heavy metals in soils, for the Danish draft soil quality standards
(85). The wisdom of using these data in this way must, however, be questioned.
(5) The ED
50
experiments conducted to date (73–77) have been relatively short term
or have required storage of amended soils under artificial conditions in the absence of
plants. This has allowed the assessment of acute effects, with direct inhibition of intracellu-
lar and extracellular enzyme activity by heavy metals. These experiments have not allowed
a realistic assessment of long-term effects, which could be quite different because, as well
as enzyme inhibition, microbial proliferation and microbial enzyme synthesis also may
be adversely impacted. This limitation could be offset by a decline in the bioavailability
of the metals with time, as they become adsorbed, chelated, and precipitated. As yet, there
are relatively few experimental data from long-term contaminated sites. Contaminated
sites, moreover, usually have the added complexity of excessive amounts of more than
one contaminant, so that it is almost impossible to assign environmental effects to any
one heavy metal or to determine dose parameters, such as ED
50

.
4. Field Studies Versus Laboratory Studies
Speir and coworkers investigated the effects of contamination of a pasture soil by Cr, Cu,
and As, acquired from runoff from a neighboring timber-treatment factory, on soil biologi-
cal properties, including enzyme activities (68,69). Heavy metal concentrations ranged
from background (Ͻ50 mg kg
Ϫ1
)toϾ1200 mg kg
Ϫ1
soil. Phosphatase, urease, and in-
vertase activities generally were considerably lower in the contaminated soil than in the
control, but differences between the lowest and highest levels of contamination (86 to
1260 mg Cr kg
Ϫ1
) did not result in further significant changes (Cr was used here as a
surrogate for all three contaminants as their concentrations correlated strongly). This im-
plies that even low levels of metal contamination could result in reduced biological activ-
ity, an effect not always observed in short-term studies. At the locations with the highest
metal concentrations, herbage yields were very low or nonexistent, possibly the cause,
and/or result, of the effect of the metals on biological activity. Sulfatase, in contrast to
the other enzymes, followed a hyperbolic relationship with increasing metal concentration
(68), falling to about 15% of the activity found in uncontaminated soil. This suggests that
this enzyme may be a good indicator of the loss of microbial activity and diminished plant
yield in contaminated sites.
Kuperman and Carreiro (70) found that soil enzyme activities declined markedly
with increasing metal concentration at a military site contaminated with As, Cd, Cr, Cu,
Pb, Ni, and Zn. Here, in contrast to the previous study (68,69), the decline in enzyme
activities generally followed a similar pattern to that of organic matter content and proba-
Copyright © 2002 Marcel Dekker, Inc.
blyindicatesaveryadverseeffectofthemetalsonsoilbiologicalactivity.Theauthors

proposedthatthedecreaseinenzymeactivitywascausedprimarilybydirectsuppres-
sionofmicrobialgrowthinthecontaminatedsoilbutconsideredthatdirectenzymeinhibi-
tionbytheheavymetalsmayalsohaveaccountedforsomeofthedecrease.Theycon-
cludedthat‘‘integrationofmicrobialbiomassandextracellularenzymeactivitymeasure-
mentsintoecologicalriskassessmentprocedureswouldpermitdirectassessmentof
negativeimpactsonthestructureandfunctionofsoilcommunitiesandecosystempro-
cesses’’(70).
Preciselyhowthegapbetweenshort-andlong-termstudies,andhowthecomplexi-
tiesofmultimetal-contaminatedsitescanbeunraveled,remainchallenges.Laboratory-
amendmentstudieshavearoleindevelopinganunderstandingoftheinteractionsofpartic-
ularheavymetalswithsoilenzymes,buttheirresultsshouldnotbeoverinterpretedor
usedinwaysthatmightleadtoerroneousormisleadingconclusions.Detaileddataon
thetoxicityofmetalsare,however,oftendifficulttoobtainfromfieldstudiesoflong-
termcontaminatedsitesbecausearelevantcontrolsoil,unlessplanned,isusuallyunavail-
able.Inaddition,agradientofmetal-contaminantconcentrationsisrarelypossible(84).
Typically,inlaboratorystudiestheresponseofmicroorganismsadaptingtoelevatedmetal
concentrationsisexamined,whereasinstudiesoflong-termmetal-pollutedsoilsthere-
sponseofmicroorganismsalreadyadaptedtoelevatedmetalconcentrationsisexamined.
Boththetypeandthesensitivityofresponsetothesedifferentformsofcontamination
maybeverydifferent,andtheresponseobtainedinthelaboratorymaybearlittlerelation
tothatseeninthefield(84).
Oneadditionalandpotentiallyseriousconcernaboutlaboratoryexperimentsusing
heavymetalsaltshasemergedfroma1999studythatshowedthatthesaltsofcationic
metals(Cd,Cr,Cu,Ni,Pb,andZn)cancausesignificantacidificationofsoilswhenadded
athighconcentrations(Ͼ10mmolkg
Ϫ1
),especiallyinsoilswithacoarsetexture(78).
Sparseattentionhasbeenpaidtothisprocessinpreviouspublications,althoughreduction
ofpHinresponsetoheavymetaladditionhasbeenobservedinsome(74,75,86,87),but
notallinvestigations(88).Itispossiblethatthisphenomenoncould,atleastinpart,

explainasignificantproportionoftheapparent‘‘inhibition’’ofsoilenzymesbyheavy
metals.Intheirinvestigation,Speiretal.(78)includedaCasalttreatmentandanacid
treatmenttodifferentiateeffectsattributabletoosmosisandacidityfromthoseattributable
toheavymetals.Theyfoundthatthelossofsoilsulfataseactivity,forallamendments,
wasalmostentirelyattributabletoacidificationofthesoilduringpreincubationbefore
assay(Fig.5).Incontrast,phosphataseactivitywasinhibitedbymetalsbutnotbyacid,
except in a coarse-textured soil in which acidification did result in loss of activity below
pH 4. They concluded that the difference in behavior of the two enzymes might be a
function of their stability to changing pH during the preincubation period. Soil sulfatase
has a narrow pH optimum (89,90), and the results indicated that activity was rapidly and
irreversibly lost at any pH significantly below this range. In contrast, the pH optimum for
phosphatase is very broad in many soils, spanning several pH units, and is probably the
resultant of a mixture of acid and alkaline phosphatases (91). It appears that this broad
pH optimum, within which the enzyme is obviously stable, had protected it from denatur-
ation during the days it was exposed to acid conditions before assay. These results have
considerable implications for the interpretation of previous studies. If the impact of soluble
and, presumably, extremely bioavailable metals is caused by pH changes, and not the
metal itself, no direct metal effect on sulfatase activity, at least in the short term, would
Copyright © 2002 Marcel Dekker, Inc.
Figure 5 Effects of amendment with heavy metals or acid on sulfatase activity in a coarse-textured
sandy soil, with percentage inhibition expressed as a function of soil pH. Amendments: Ca, —᭜—;
Cd, —᭡—; Cu, —᭹—; Pb —᭛—; Zn —ᮀ—; Ni —■—; Cr —᭝—; Acid—᭺—. (Adapted,
with permission, from Ref. 78.)
be discernible at most metal-contaminated sites or after materials, such as sewage sludge
had been applied to land.
C. Heavy Metals in Sewage Sludge
Land application of sewage sludge can result in the contamination of agricultural soils
by heavy metals. Although this practice returns valuable nutrients and organic matter to
the land, it is controlled in many countries by rules and guidelines stipulating maximal
contaminant loading of the sludge, maximal annual metal application rates, and maximal

soil metal-concentrations. Assessment of the impacts of sewage sludge–borne heavy met-
als on soil microorganisms and microbial processes is complicated by the high organic
matter and nutrient contents of the sludge. Frankenberger et al. (92) found that soil urease
activity was inhibited at low sludge-loading rates but markedly enhanced at higher rates.
This initial reduction was attributed to the extremely high contaminant load in the sludges
investigated, and the enhancement to the sludge organic matter and nutrients stimulat-
ing microbial activity and urease synthesis. Other studies, using less contaminated
sludges, also have demonstrated that enzyme activities increased steadily with increasing
rates of sludge application (93–95). However, in one of these studies, sewage sludge
stimulated activity to a lesser extent than other organic amendments, possibly because of
its metal content (94). Long-term studies are required, preferably at sites where sludge
application has long ceased and the sludge nutrients and readily degradable organic matter
have gone. Currently, some of the most important research on the effects of heavy metals
Copyright © 2002 Marcel Dekker, Inc.
in sewage sludge is emerging from long-term sites in the United Kingdom and Germany
(reviewed in 83–85,96), but very little enzymological analysis has been conducted on
their soils. Other contaminated sites, such as those investigated by Tyler (66,67), Speir
et al. (68,69), and Kuperman and Carreiro (70), especially on agricultural soils, may,
however, be considered as surrogates for land that had previously had sewage sludge
applied.
D. Pesticides
There are large numbers of pesticides currently registered for agricultural use throughout
the world and many that no longer are used but whose residues and metabolites still are
found in agricultural soils. All of these have been subjected to more or less intensive
investigation for the determination of potential side effects on soil organisms and biologi-
cal processes. Arising from this work, soil biological criteria have been established for
the routine assessment of pesticides for registration purposes (e.g., 97–100). However,
these do not include assessments of impacts on soil enzyme activities, even though there
have been a large number of investigations of pesticide–soil enzyme interactions (exten-
sively reviewed in 6,10,101). One reason for this is readily apparent on reading the com-

prehensive review of Scha
¨
ffer (10); there are very few consistent patterns of soil enzyme
inhibition by pesticides, even at concentrations greatly exceeding recommended applica-
tion rates. Another reason is that no attempts have been made, or could have been made,
to evaluate the consequences for biodiversity, biological activity, and/or soil fertility of
enzyme inhibition when it did occur. The literature on this topic is very similar to that
describing the effects on soil enzymes of agricultural practices such as fertilizer applica-
tion, use of other agrochemicals, and land management; pesticides inhibit/have no effect
on/activate soil enzymes, the particular response depending on factors such as soil type
and experimental design. For example, long-term field application of both the amine and
ester formulations of the herbicide 2,4-D at normal agronomic rates, in two separate stud-
ies, led to the following conclusions: ‘‘The effects of long-term 2,4-D application (both
ester and amine) were neither ecologically significant nor did they interfere with nutrient
cycling’’ (102); ‘‘dehydrogenase activity and soil microbial respiration were reduced sub-
stantially by the ester, indicating that the ester probably interfered with nutrient cycling’’
(103). In the former study, urease and acid- and alkaline-phosphatase activities were tem-
porarily reduced, whereas in the latter, urease activity was permanently depressed.
Pesticides are not designed to inhibit soil enzymes. Some may indeed be enzyme
inhibitors, but their structural diversity makes it impossible to foresee direct negative ef-
fects on the hydrolase activities we measure by using artificial substrates and assay condi-
tions. They still could have a negative effect on soil enzyme activity; obviously a fungicide
or a herbicide will reduce enzyme activity, if not by direct inhibition, then by removal of
a source of soil enzymes (104). However, in most instances, any depressive effect has
been found to disappear after several days or weeks (10) and may be replaced by enhanced
enzyme activity, possibly from enzymes released from lysed cells (105) or as a response
to a flush of microbial activity as the killed organisms are metabolized. Recovery of soil
biological activity some time after application is the basis for an ecological approach for
the assessment of side effects of agrochemicals on soil microorganisms proposed by
Domsch et al. (106). From a review of a large number of case studies, side effects were

compared with the depressive effects resulting from natural stress factors, such as fluctua-
tions of temperature, water content, pH, and physical disturbances. Such natural stresses
Copyright © 2002 Marcel Dekker, Inc.
were found to cause depressions of some properties by up to 90%, but the depressions
were short-lived, and recovery phases averaged 18 days. On this basis, it was proposed
that reversible side effects of agrochemicals causing delays of recovery of microbiological
properties of up to 30 days are normal, those resulting in delays of 60 days are tolerable,
but those with delays of more than 60 days may be critical. A minimal monitoring period
of 30 days for the detection of persistent side effects was proposed. On this basis, the
studies reviewed by Scha
¨
ffer (10) and subsequent investigations (e.g., 105,107–111) sug-
gest that the depressions of soil hydrolytic enzyme activities almost all fall into the ‘‘nor-
mal’’ category. This reinforces the conclusions of Ladd (112) that pesticide applications
have little or no effect on enzyme activity in soils.
Is it, therefore, worthwhile to continue to test for pesticide effects on soil enzyme
activities? The answer depends upon the context of the research. Existing pesticides have
been studied extensively at a range of concentrations in different soil types. Further investi-
gations may be profitable only if they concentrate on degradative enzymes from the per-
spective of understanding mechanisms of pesticide metabolism in soils and/or for the
cleanup of contaminated soils. On balance, it probably is important that newly registered
pesticides continue to be subjected to tests for effects on soil enzymes, even though they
have already passed registration criteria that are arguably more stringent than these tests.
It is conceivable that a new chemical or a metabolite may, by design or accident, be a
particularly potent inhibitor of a soil enzyme. If the inhibition extends beyond 60 days,
then according to the definition of Domsch et al. (106), a critical situation may exist and
further investigation is warranted.
VI. CONCLUSIONS
There have been many studies of the impacts of soil physical and chemical degradation
on soil hydrolase enzyme activities and attempts made to use enzyme activity measure-

ments to determine or predict the extent and/or rate of soil degradation or recovery after
degradation. The major barriers to progress arise from the limitations of our methodology
and, therefore, our understanding of the different compartments of soil enzyme activity
and their roles in soil fertility and productivity.
When soils have been subjected to physical degradation comprising loss of surface
materials by erosion or mining or subjected to intensive cropping, the primary effect is
loss of organic matter. Loss of enzyme activity is a consequence because there is always
a strong correlation between these properties. Generally, the assay of soil enzyme activities
does not provide any more information about potential soil degradation from such physical
causes than does the measurement of organic C alone, nor does it give any information
about the short-term productivity of the soil. However, techniques that provide insight
into the various compartments of enzyme activity may provide information on soil organic
matter quality that could be useful in assessing the extent of soil degradation.
In the early stages of recovery of degraded soils, or development of ‘‘technogenic’’
soils, soil enzyme activities correlate closely with plant yields and increase more rapidly
than does soil organic C content. In these situations, the enzymes can be used as indicators,
or biomarkers, of progress toward recovery and possibly predictors of how long recovery
will take. Some enzymes, e.g., the carbohydrases, seem to be better predictors than others,
but because we do not know how soil, plant, and environmental factors influence the
recovery of individual enzymes, this predictive role is still only an experimental tool.
Copyright © 2002 Marcel Dekker, Inc.
Soil enzyme activities are not particularly sensitive to contamination by oil and oil
products; there is little adverse effect at concentrations of crude oil sufficient to kill all
plant life. Of the fuel products tested, only the aromatic compounds, and possibly only
benzene, significantly inhibited soil enzyme activity.
Soil characteristics provide considerable ‘‘protection’’ of soil enzymes from heavy
metals. Because these same characteristics also determine the ‘‘bioavailability’’ of metals
to plants and soil organisms, soil enzymes could have a role as ecotoxicological indicators
or biosensors of environmental effects. Dose–response curves have been used to determine
the impact of heavy metal salts on soil enzyme activities, but because different enzymes

show different levels of inhibition, even within a single soil, it is not possible to determine
what level is acceptable. Enzyme inhibition data from metal-salt amendment studies have
been used to support soil quality criteria for heavy metals. It is, however, questionable
whether this is appropriate, considering that metals rarely enter soils in this form or reach
such high concentrations with a single application. Metal-salt amendment experiments
have also generally been short-term studies that assess acute, mainly direct-inhibition,
effects on soil enzyme activities. The response in long-term metal-polluted soils is likely
to be considerably different, and effects on soil enzyme activities also may include those
attributable to reduced enzyme synthesis and impaired microbial growth and activity.
One form of long-term contamination that has received a great deal of attention is
that arising from the application of sewage sludge to land. However, the few studies that
have included enzyme assays generally found that the enhancement of overall biological
activity resulting from the readily available C and nutrients masked any negative impact
on the enzyme activities. There are few instances of significant and prolonged adverse
effects of pesticides on soil enzyme activities.
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